Feed for improving water quality and application thereof in soft-shelled turtle culture

Abstract

The invention provides a feed capable of improving water quality and application of the feed in soft-shelled turtle breeding, the feed comprises a composite microecological preparation prepared from chitosan oligosaccharide and Weissella sinus, and the soft-shelled turtle breeding method is characterized in that soft-shelled turtles are fed by a method of splashing the feed into a water body, and the water flora simultaneously decomposes the feed and the waste digested by the soft-shelled turtles to improve the water quality. A water quality analysis result of a soft-shelled turtle culture water body shows that after the compound micro-ecological preparation is added into the feed, the ammonia nitrogen content in the water body is remarkably reduced, the dissolved oxygen content is relatively high, the water quality is acidic, meanwhile, the abundance of beneficial bacteria in the water body is increased, the community diversity is reduced, and the flora balance in the water body is maintained.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and in particular relates to a feed for improving water quality and application of the feed in turtle breeding. Background Art

[0002] Soft-shelled turtles are also known as water fish, round fish and turtles. They are oviparous reptiles that live on land and in water. Soft-shelled turtle meat is delicious and nutritious. It has the effects of clearing away heat and nourishing yin, calming the liver and extinguishing wind, and softening and dispersing lumps. It is not only a delicious dish on the table, but also a widely used tonic medicine and Chinese medicine material. At present, in the process of artificial breeding of soft-shelled turtles, the nutrients provided cannot meet the growth needs of soft-shelled turtles, and the nutrients taken by soft-shelled turtles cannot meet their actual needs, resulting in long-term imbalance of the intestinal flora of soft-shelled turtles, weak immunity, low survival rate, and poor muscle quality of adult soft-shelled turtles.

[0003] Microecological preparations are composed of a variety of beneficial bacteria, have no toxic side effects, and no drug residues. They are green water quality improvers and additives that can significantly improve the breeding ecological environment, enhance the disease resistance of soft-shelled turtles, and improve the quality of soft-shelled turtles. They have been accepted by more and more breeders. However, in actual application, due to differences in product characteristics and production processes, these biological preparations have unsatisfactory effects in practical application, poor stability, and obvious differentiation.

[0004] Patent document CN118160814A discloses a composite microecological preparation and its application in juvenile soft-shelled turtle breeding, wherein the composite microecological preparation comprises a variety of probiotics including lactic acid bacteria, Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis, Chinese herbal extracts, adjuvants and nutrients. Through the synergistic effect of probiotics and Chinese herbal extracts, the degradation capacity of organic matter, the improvement effect of water quality in aquaculture water bodies and the feed utilization rate of juvenile soft-shelled turtles are effectively improved, but there is still a lack of research on regulating the intestinal flora of soft-shelled turtles and improving the survival rate of soft-shelled turtles.

[0005] Therefore, providing a preparation or method that can effectively improve the water quality of turtle breeding water and thus promote the growth of turtles is of great significance to the turtle breeding industry. Summary of the invention

[0006] In order to solve the technical problems existing in the current turtle breeding process, the present invention provides a feed for improving water quality and its application in turtle breeding. The feed includes a composite microecological preparation prepared from chitosan oligosaccharides and Weissella sinusoids. The use of the feed can not only effectively improve the digestion and absorption capacity and immunity of the turtle, but also improve the water quality conditions for the turtle's survival.

[0007] On the one hand, the present invention provides a composite microecological preparation, comprising Weissella sinensis, wherein the preservation number of Weissella sinensis is CGMCC NO.32640.

[0008] The increase in aquaculture density has increased the risk of disease outbreaks. Common diseases in aquaculture include bacterial sepsis caused by pathogenic bacteria such as Aeromonas hydrophila. Aeromonas hydrophila is susceptible to infection of both aquatic animals and humans, and is highly dangerous in the aquaculture industry. Due to the abuse of drugs, the growth environment of aquatic organisms has been polluted and the animals themselves have developed drug resistance. Therefore, it is urgent to find and develop drug substitutes to improve the resistance of aquatic animals to intestinal pathogens and the quality of the water environment in which they grow.

[0009] Antagonism is one of the ways in which bacteria or other microorganisms interact with each other. Using strains with strong antagonistic properties to inhibit the growth, reproduction or pathogenicity of pathogens is one of the means of biological control. The present invention utilizes the inhibitory effect on Aeromonas hydrophila to screen three probiotics, Weissella cibaria, Enterococcus faecalis, and Lactococcus lactis, from the intestinal tract of turtles by dilution coating plate method. Further, the three probiotics were co-cultured with Aeromonas hydrophila using the Oxford cup method, and it was found that Weissella cibaria had the strongest inhibitory effect on Aeromonas hydrophila. In addition, the present invention has proved through experiments that the composite microecological preparation or turtle breeding feed prepared from Weissella cibaria obtained from the intestinal tract of turtles can significantly improve the antioxidant capacity of turtles and the quality of turtle breeding water after feeding turtles.

[0010] The Weissella sinusoidalis provided by the present invention was screened from the intestines of soft-shelled turtles and was deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China National Microbiological Culture Collection Committee on November 15, 2024, Beijing, China, with a deposit number of CGMCC NO.32640.

[0011] The bacterial flora structure in the turtle's intestine was then analyzed. The results showed that adding Weissella sinusoids alone to the feed could increase the diversity of the turtle's intestinal flora, and the intestine was enriched with three bacterial genera: Turicibacter, Pediococcus and Weissella; and after feeding with feed supplemented with complex microecological preparations, the diversity of the turtle's intestinal flora structure was significantly improved, and the two bacterial genera Paeniclostridium and Cellulosilyticum were enriched in the turtle's intestine. These two genera can promote protein decomposition and cellulose degradation, which can be more conducive to the conversion of nutrients.

[0012] Furthermore, the composite microecological preparation also includes any one or more prebiotics selected from the group consisting of oligofructose, oligochitosan, and β-glucan.

[0013] Prebiotics are indigestible ingredients in food or feed that can selectively stimulate the growth of beneficial bacteria in the digestive tract of host animals, thereby having a beneficial effect on animals, including oligosaccharides, microalgae and natural plants. In recent years, prebiotics have shown the efficacy of preventing and treating diseases in aquaculture. Various types of prebiotics, including oligoxylose and oligogalactose, have been used as alternatives to antibiotics to promote intestinal health and prevent diseases by selectively promoting the growth of beneficial bacteria. Chitooligosaccharide is a natural alkaline polymer of glucosamine. It is an oligosaccharide product with a degree of polymerization between 2 and 20 obtained by degrading chitosan by special bioenzyme technology. The molecular weight is ≤3200Da. It is a low-molecular-weight product with good water solubility, great functional effects and high biological activity. It has many unique functions that chitosan does not have, such as high solubility, full solubility in water, and easy absorption and utilization by organisms. Due to its beneficial functions in regulating aquatic animal immunity, promoting growth and anti-oxidation reactions, it is used as a feed additive, vaccine adjuvant and aquatic product preservative for aquatic animals. Another prebiotic used as an immunomodulator and antioxidant in aquaculture is β-glucan. Adding β-glucan to the feed can improve the immunity and antioxidant index of soft-shelled turtles. At the same time, there is another prebiotic, oligofructose, which can also maintain the balance of intestinal flora and enhance the body's immunity. Oligofructose, also known as oligofructose or sucrose oligosaccharide, is divided into sucrose sources (beets, sugar cane, etc.) and inulin sources (chicory, Jerusalem artichoke). Oligofructose is a natural active substance with a sweetness of 0.3 to 0.6 times that of sucrose. It not only maintains the pure sweetness of sucrose, but also tastes lighter than sucrose. It is a new sweetener with the functions of regulating intestinal flora, proliferating bifidobacteria, and immunomodulation. It is hailed as the most promising new generation of additives after the era of antibiotics.

[0014] The present invention further adds different prebiotics to the feed for separate feeding comparison. The results of α-diversity analysis show that the α-diversity of the COS group (chitosan oligosaccharide) is slightly higher than that of the FOS group (fructogenic saccharide), but there is no significant difference; β-diversity analysis shows that after the two groups of prebiotics are fed, there is a significant difference in the intestinal flora structure of the soft-shelled turtle compared with the control. The β-diversity dispersion of the COS group (chitosan oligosaccharide) is slightly lower than that of the FOS group (fructogenic saccharide), and the ability to regulate the flora structure is stronger; in addition, in the chitosan oligosaccharide group, the contents of Cellulosilyticum, Helicobacter and Solibacillus are higher, indicating that adding chitosan oligosaccharide or fructogenic saccharide to the feed can change the composition of the intestinal microbiome of the soft-shelled turtle and differentially enrich specific bacteria.

[0015] Furthermore, the prebiotic is chitosan oligosaccharide.

[0016] According to the comparison results after adding a separate component to feed the turtle, it can be seen that different prebiotics or probiotics have different effects on improving the antioxidant and antibacterial capabilities of the turtle, so the promotion of the composite microecological preparations prepared by different prebiotics or probiotics may also be different. The present invention proves through comparative experiments that the addition of a composite microecological preparation prepared with chitosan oligosaccharides and Weissella edodes in feed significantly improves the total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) enzyme activity, while reducing the acid phosphatase (ACP) and MDA (malondialdehyde) levels, while other groups have no significant effect on these index levels, and the composite microecological preparation prepared by chitosan oligosaccharides and Weissella edodes is preferred.

[0017] Furthermore, the content of Weissella sinusoidalis is 1×10 7 ~1×10 9 CFU / g, and the content of chitosan oligosaccharide is 0.1-0.5wt%.

[0018] In some embodiments, the content of Weissella sinusoidalis is 1×10 7 ~1×10 8 CFU / g, and the content of chitosan oligosaccharide is 0.1-0.3wt%.

[0019] In some embodiments, the content of Weissella sinusoidalis is 1×10 8 CFU / g, and the content of chitosan oligosaccharide is 0.3wt%.

[0020] On the other hand, the present invention provides a turtle feeding feed, comprising the composite microecological preparation as described above.

[0021] The composite microecological preparation provided by the present invention can be added with compatible ingredients as needed, such as fish meal, rice bran, bran, bean cake, adhesives, additives, vitamins, inorganic salts, etc., within the scope of not damaging the effects of the present invention. Various feeds or products with the same effects prepared on the basis of the present invention are within the protection scope of the present invention.

[0022] In another aspect, the present invention provides a method for breeding soft-shelled turtles, using the feed as described above for breeding.

[0023] In another aspect, the present invention provides a use of a composition for preparing an agent for regulating the balance of intestinal flora of soft-shelled turtles, wherein the composition comprises Weissella sinusoidalis and chitosan oligosaccharide.

[0024] Studies have shown that the composition of intestinal flora is different after adding different prebiotics or probiotics or compound microecological preparations. The colony diversity of the probiotic group is significantly higher than that of the prebiotic group. The compound microecological preparation group prepared by Weissella sinusoids probiotics and chitosan oligosaccharide prebiotics is enriched with bacterial genera that are beneficial to the transformation of nutrients and maintains the balance of the flora.

[0025] In another aspect, the present invention provides a use of a composition for preparing an agent for improving the immunity of soft-shelled turtles, wherein the composition comprises Weissella sinensis and chitosan oligosaccharide.

[0026] Furthermore, through the analysis of antioxidant indices and nonspecific immune indices in the liver tissue of soft-shelled turtles, the present invention found that the activity of catalase (CAT) in the chitosan oligosaccharide group was significantly higher than that in the oligofructose group, indicating that chitosan oligosaccharides are superior to oligofructose in improving antioxidant capacity. At the same time, the superoxide dismutase (SOD) in the composite probiotic preparation group was significantly higher than that in the chitosan oligosaccharide group, indicating that the composite probiotic preparation is better than chitosan oligosaccharide in improving antioxidant capacity. At the same time, the composite probiotic preparation significantly reduced the levels of acid phosphatase (ACP) and malondialdehyde (MDA), and improved the antibacterial effect of soft-shelled turtles, indicating that adding composite probiotic preparations to feed can significantly improve the immunity of soft-shelled turtles.

[0027] In another aspect, the present invention provides a use of a composition for preparing an agent for promoting digestion and absorption of nutrients, wherein the composition comprises Weissella sinensis and chitosan oligosaccharide.

[0028] Furthermore, the results of intestinal digestive enzyme activity analysis showed that Weissella sinusitis, Enterococcus faecalis and compound microecological preparations significantly increased the activity of trypsin, among which Weissella sinusitis had the strongest effect; chitosan oligosaccharides increased the activity of lipase and amylase, and the effect was better than oligofructose. At the same time, after adding chitosan oligosaccharides or compound microecological preparations to the feed, the number and length of the turtle's colon villi increased sharply, indicating that the use of compound microecological preparations containing Weissella sinusitis and chitosan oligosaccharides can promote the digestion and absorption of nutrients in the turtle's intestines.

[0029] In another aspect, the present invention provides a use of a composition for preparing an agent for increasing the amino acid content of soft-shelled turtle muscle, wherein the composition comprises Weissella sinensis and chitosan oligosaccharide.

[0030] Studies have shown that adding the prebiotics, probiotics or composite microecological preparations provided by the present invention to feed can increase the content of umami amino acids in turtle muscle, and adding the composite microecological preparations to feed significantly increases the content of glutamate, aspartic acid and glycine.

[0031] In another aspect, the present invention provides a feed for improving the water quality of turtle breeding water, comprising Weissella sinusophila, and the preservation number of Weissella sinusophila is CGMCC NO.32640.

[0032] Furthermore, the feed also includes any one or more prebiotics selected from the group consisting of oligofructose, oligochitosan, and β-glucan.

[0033] Furthermore, the prebiotic is chitosan oligosaccharide.

[0034] Furthermore, the content of Weissella sinusoidalis is 1×10 7 ~1×10 9 CFU / g, and the content of chitosan oligosaccharide is 0.1-0.5wt%.

[0035] On the other hand, the present invention provides a method for improving water quality when breeding soft-shelled turtles, using the feed as described above to feed the soft-shelled turtles.

[0036] Furthermore, the feed is fed to the soft-shelled turtle by spraying, and the feed enters the water body while being fed to the soft-shelled turtle. The water body flora decomposes the feed, and the probiotics or prebiotics in the feed regulate the water body flora to improve the water quality.

[0037] On the other hand, the present invention provides a use of a feed for preparing an agent for reducing the content of ammonia nitrogen, total phosphorus and chemical oxygen demand in turtle breeding water, wherein the feed comprises Weissella sinusophila, and the preservation number of the Weissella sinusophila is CGMCC NO.32640.

[0038] In another aspect, the present invention provides a use of a feed for preparing an agent for increasing the dissolved oxygen content in turtle breeding water, wherein the feed comprises Weissella sinusoidalis, and the preservation number of the Weissella sinusoidalis is CGMCC NO.32640.

[0039] In another aspect, the present invention provides a use of a feed for preparing an agent for maintaining the balance of flora in turtle breeding water, wherein the feed comprises Weissella sinusophila, and the preservation number of the Weissella sinusophila is CGMCC NO.32640.

[0040] Because the compound microecological preparation is mixed in the feed and then fed to the turtle, part of the feed will enter the water body at the same time as it enters the turtle's intestine; and because the feed eaten by the turtle will be excreted into the water body like the intestinal flora, the intestinal flora and water flora of the turtle are also affected by the fed bacterial agents. After some flora in the water body decompose the food residues or feces of the turtle, ammonia nitrogen will be produced, which will then have a stressful effect on the growth of the turtle. Therefore, the fed bacterial agent needs to inhibit the abundance of ammonia nitrogen-producing colonies. The verification results show that after adding the compound microecological preparation to feed the turtle, the dissolved oxygen in the water where the turtle grows is high, the content of ammonia nitrogen and sulfide is reduced, the water is slightly acidic, the water quality is better, and at the same time, the abundance of beneficial bacteria in the water body increases, and the abundance of harmful bacteria Proteobacteria is reduced, maintaining the colony balance of the aquaculture water body.

[0041] Furthermore, the feed also includes any one or more prebiotics selected from the group consisting of fructooligosaccharides, chitosan oligosaccharides, and β-glucan.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. After feeding the soft-shelled turtle with the feed provided by the present invention, the balance of the soft-shelled turtle's intestinal flora is regulated, and the soft-shelled turtle's immune ability and the soft-shelled turtle's intestinal digestion and absorption capacity of nutrients are improved;

[0044] 2. Adding compound microecological preparations to feed regulates the abundance of bacteria in the water and increases the content of beneficial bacteria;

[0045] 3. After feeding the soft-shelled turtle with feed, the ammonia nitrogen content in the breeding water is significantly reduced, the dissolved oxygen content is higher, and the water quality is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0047] Figure 1 This is a diagram of the probiotic culture in the laboratory of the present invention;

[0048] Figure 2 This is the structure and composition diagram of the intestinal flora analyzed by β-diversity analysis of the present invention

[0049] Figure 3 The super index (A) and Shannon index diagram (B) of the intestinal flora α-diversity analysis of the present invention are shown;

[0050] Figure 4 This is a diagram of the structure of intestinal flora analyzed at the phylum level of the present invention;

[0051] Figure 5 This is a genus level analysis of the intestinal flora map of the present invention;

[0052] Figure 6 This is a comparative analysis diagram of multiple groups of intestinal flora of the present invention;

[0053] Figure 7 This is the LefSe score diagram of the intestinal flora of the present invention;

[0054] Figure 8 The anatomical structure diagram of the intestinal tissue of the turtle in the present invention;

[0055] Fig. 9 The effect of the present invention on the antioxidant capacity and immune-related enzyme activity of turtle liver;

[0056] Fig.10 This is a diagram of the digestive enzyme activity of the turtle intestine in the present invention;

[0057] Fig.11 The crude protein content (A), essential amino acid content (B), umami amino acid content (C), glutamic acid content (D), glycine content (E), aspartic acid content (F) and alanine content (G) in the soft-shelled turtle muscle of the present invention are analyzed;

[0058] Fig.12 The chemical oxygen demand (A), nitrite nitrogen content (B), sulfide content (C), ammonia nitrogen content (D), dissolved oxygen content (E) and pH (F) analysis results of the water body in the present invention;

[0059] Fig.13 This is a diagram of the composition of the water flora structure for the β-diversity analysis of the present invention;

[0060] Fig.14 This is the super index diagram of water flora for α-diversity analysis of the present invention;

[0061] Fig.15 It is the water body flora map analyzed at the phylum level of the present invention;

[0062] Fig.16 This is a genus level analysis of water flora map of the present invention;

[0063] Fig.17 This is a comparative analysis diagram of multiple groups of water quality flora of the present invention;

[0064] Fig.18 This is the LefSe diagram for the differential analysis of water quality bacterial flora of the present invention. DETAILED DESCRIPTION

[0065] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0066] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0067] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0068] Example 1: Preparation of feed using the composite microecological preparation provided by the present invention

[0069] 1. Preparation of Weissella sinensis bacterial suspension

[0070] The Weissella sinusoidalis liquid in the cryopreserved tube was inoculated into MRS liquid culture medium at a 2% inoculation rate, cultured in a 37°C incubator for 24 hours, and subcultured twice to obtain a fresh bacterial liquid; a portion of the fresh bacterial liquid was taken, centrifuged at 5000r / min for 5 minutes, and the supernatant was gently aspirated into another sterile centrifuge tube to obtain the bacterial liquid supernatant; the centrifuged bacteria were washed twice with sterile PBS, and then the strain OD600nm was adjusted to 1.0 using PBS buffer to obtain a bacterial resuspension. Then, 10% dHO was added per gram of the basic diet (purchased from Zhejiang Jindadi Agricultural Technology Co., Ltd.) 8 The colony count of CFU was calculated by weighing the bacterial resuspension. The basic diet formula is shown in Table 1.

[0071] Table 1 Nutritional composition of the basic diet

[0072]

[0073] 2. Weigh chitosan oligosaccharide powder (purchased from Zhejiang Jinke Pharmaceutical Co., Ltd., model SLKG) according to the addition ratio of 0.003g chitosan oligosaccharide per gram of basic diet, that is, 0.3wt%;

[0074] 3. Add the bacterial solution and chitosan oligosaccharide powder to the basic diet and mix well before use.

[0075] Example 2: Screening and separation of probiotics

[0076] Through the dilution plate method, many different strains of bacteria were screened from the intestines of healthy soft-shelled turtles, and through the Oxford cup method, three strains were selected that had a significant inhibitory effect on Aeromonas hydrophila, a common pathogenic bacteria of soft-shelled turtles. They are Weissella cibaria, Enterococcus faecalis, and Lactococcus lactis.

[0077] The specific steps of the Oxford cup method for screening three types of probiotics are as follows:

[0078] (1) Prepare nutrient agar medium (NA)

[0079] Prepare NA plates and MRS plates in advance and dry them. The NA and MRS culture media were purchased from Qingdao Hibo Biotechnology.

[0080] (2) Preparation of Aeromonas hydrophila bacterial solution

[0081] Activate Aeromonas hydrophila (strain source: isolated from the intestine of diseased turtle in our laboratory earlier) by streaking on NA agar plates, then pick a single colony into NB medium, culture it aerobically at 37°C for 24 h, then transfer it to new NB medium at a ratio of 2%, culture it aerobically at 37°C for 24 h to obtain fresh bacterial liquid.

[0082] (3) Preparation of fermentation broth of three probiotic strains

[0083] The probiotic liquid in the cryopreserved tube was inoculated into MRS liquid culture medium at a 2% inoculation rate, and cultured in a 37°C incubator for 24 hours. After subculturing twice, a fresh liquid was obtained; a portion of the fresh liquid was taken, centrifuged at 5000r / min for 5 minutes, and the supernatant was gently aspirated into another sterile centrifuge tube to obtain the supernatant of the liquid; the centrifuged bacteria were washed twice with sterile PBS, and then the strain OD was measured by continuing to use PBS buffer. 600 nm was adjusted to 1.0 to obtain a heavy suspension of bacterial cells.

[0084] (4) Oxford cup antibacterial experiment

[0085] Prepare NA culture medium and sterilize it; after the temperature drops below 47°C, add Aeromonas hydrophila bacterial solution and shake well; pour it onto a plate, put an Oxford cup on it after solidification, add 100 μL of probiotic fermentation broth, supernatant after centrifugation of the fermentation broth, bacterial resuspension, and PBS (blank control) into the wells respectively, culture at 37°C for 24 hours, and observe whether there is an inhibition zone.

[0086] turn out( Figure 1), Weissella sinusoidalis has the strongest inhibitory effect on Aeromonas hydrophila. The Weissella sinusoidalis screened from the intestinal tract of the soft-shelled turtle was deposited in the China General Microbiological Culture Collection Center (CGMCC) on November 15, 2024, with a deposit number of CGMCC NO.32640.

[0087] Example 3: Analysis of intestinal microbial composition and diversity

[0088] According to the characteristics that prebiotics and probiotics can maintain the balance of intestinal flora and improve the body's immune ability, this example further explores the effects of prebiotics, probiotics and the composite microecological preparation provided by the present invention on the diversity of intestinal flora.

[0089] Eight rice fields (about 40m×10m in area, each rice field was divided into three small fields on average) were selected for the experiment. 36-month-old Chinese soft-shelled turtles were randomly divided into 8 groups (6 experimental groups + 2 control groups), with an initial average body weight of 1±0.2kg and an average shell length of 20.2cm. They were fed with a designated diet for 4 weeks. The ingredients and dosages added to the feeds of the six experimental groups are shown in Table 2. The control group included a basic diet and a commercially available bacterial agent (purchased from Zhejiang Hemudu Biotechnology Co., Ltd.). The dietary formula of the basic feed and its approximate composition are shown in Table 1. The soft-shelled turtles were fed twice a day (8:30, 17:30). The sampling time point was selected 2 weeks after feeding. Six soft-shelled turtles were randomly selected from each group (two were randomly selected from each small rice field) and their body weights were measured. Jugular vein blood samples were collected and centrifuged at 4℃ (3500×g) for 15min to obtain serum samples. Hind leg muscle tissue, liver tissue, intestinal tissue and colon contents were collected and frozen with liquid nitrogen.

[0090] The intestinal flora in the intestinal contents were sequenced and aligned using 16S rRNA gene sequencing, and the α-diversity index, including Chao1 and Shannon index, was calculated using QIIME2 to evaluate the complexity of the microbial community (21). β-diversity analysis was performed to investigate the differences within and between groups based on principal coordinate analysis (PCoA) with the R vegan package. In addition, microbial function prediction was achieved using PICRUSt2. Functional pathway annotation was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. All data processing and analysis were performed using OmicShare tools (https: / / www.omicshare.com / tools).

[0091] Table 2 Ingredients and dosage added in different groups

[0092]

[0093] β-diversity analysis showed that ( Figure 2 ), compared with the control group, the intestinal flora structure of the WC group (Weissella sinusitis) and the CWC group (compound microecological preparation) was significantly different, indicating that the intestinal flora structure changed after feeding the compound microecological preparation; the α-diversity of the COS group (chitosan oligosaccharide) was slightly higher than that of the FOS group (oligofructose), but there was no significant difference and the β-diversity dispersion was slightly lower, indicating that the intestinal flora abundance of the COS group was higher than that of the FOS group and the flora difference within the group was smaller. α-diversity analysis ( Figure 3 ) found that the α-diversity of the WC group and the CWC group was slightly higher than that of the control group, but there was no significant difference. The above results indicate that the intestinal flora structure of the soft-shelled turtle is more stable after feeding with the compound probiotic preparation.

[0094] By analyzing the intestinal flora at the phylum level ( Figure 4 ) found that the COS group and FOS group had higher Firmicutes / Bacteriota values ​​than the control group, indicating that both chitosan oligosaccharides and oligofructose improved the turtle's ability to absorb nutrients and store energy, and chitosan oligosaccharides had a better effect; analysis of the intestinal flora genus level ( Figure 5 ) found that COS and FOS groups were enriched with more Cetobacterium genus compared with the control group. The increase of Cetobacterium can increase the synthesis of vitamin B12, which is very important for the body's normal nerve function, red blood cell formation and other physiological processes. WC and CWC groups were enriched with more Romboutsia genus compared with the control group. Romboutsia genus can help decompose food, promote nutrient absorption, assist the body to produce some short-chain fatty acids that are beneficial to the intestine, provide energy for intestinal cells, and maintain the health of intestinal mucosa.

[0095] The results of difference analysis ( Figure 6 and Figure 7 ) showed that the bacterial composition structure of different experimental groups was different. The intestines of the COS group were enriched with Dielma, the intestines of the WC group were enriched with Turicibacter, Pediococcus and Weissella, and the CWC group was enriched with Paeniclostridium and Cellulosilyticum. Paeniclostridium and Cellulosilyticum can promote protein decomposition and cellulose degradation, which is more conducive to the conversion of nutrients. This shows that adding Weissella sinusoids or compound microecological preparations to the feed can significantly improve the diversity of intestinal flora and maintain the balance of intestinal flora.

[0096] Example 4: Intestinal morphology analysis

[0097] In order to further study the changes in the intestinal morphology of the 8 groups of turtles after feeding shown in Table 2 in Example 3, this example conducted tissue morphology observations on the intestinal tissues of the turtles collected in different groups after feeding in Example 3. The colon tissue was washed with phosphate buffered saline (PBS) and quickly fixed in 4% paraformaldehyde. It was paraffin embedded after 24 hours. The slices were sliced ​​longitudinally to a thickness of 4 μm, dehydrated using an automatic dehydrator, and stained with hematoxylin-eosin (H&E). Histomorphology was observed using a digital pathology scanner (Kfbio, China) and an optical microscope (Nikon, Japan).

[0098] like Figure 8 As shown in the data, after adding chitosan oligosaccharides and compound probiotic preparations to the feed, the number and length of the turtle's colon villi increased sharply, and the effect of chitosan oligosaccharides was stronger than that of the compound probiotic preparations; while the length of the intestinal villi with added oligofructose decreased sharply, there was no significant difference in the intestinal villi of the Enterococcus faecalis and Weissella sinusoids groups, and the length of the intestinal villi of Lactococcus lactis increased. The above results indicate that adding chitosan oligosaccharides or compound probiotic preparations to the feed can significantly improve the digestion and absorption of nutrients by the turtle, thereby promoting the growth of the turtle.

[0099] Example 5: Immunoenzyme activity analysis

[0100] This example further analyzes immune indexes of the turtle liver tissues collected from different groups after feeding in Example 3. Five indicators of antioxidant action in the liver were detected using a commercial kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions, namely: total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) levels. T-AOC activity and GSH-PX activity were determined by colorimetry, CAT activity was determined by visible light method, SOD activity was determined by WST-1 method, and MDA content was determined by thiobarbituric acid (TBA) method. In addition, three indicators of nonspecific immunity were also determined, including liver lysozyme (LZM), alkaline phosphatase (ALP) and acid phosphatase (ACP) levels. LZM activity was determined by turbidity, and ALP and ACP activities were determined by microenzyme labeling method. All reactions were carried out in 96-well plates, and absorbance was measured using a microplate reader (Thermo Scientific TM 5580, China) read.

[0101] The production of MDA will cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, and is cytotoxic. Excessive levels of MDA will be harmful to the body. CAT is a reactive oxygen scavenger that can decompose reactive oxygen species such as hydrogen peroxide produced during the body's metabolism. At the same time, SOD is an antioxidant metal enzyme in the body that can catalyze the dismutation of superoxide anion radicals to generate oxygen and hydrogen peroxide, playing a vital role in the body's oxidation and antioxidant balance. Therefore, the increase in the activity of CAT and SOD indicates that the body's antioxidant capacity has been improved. The analysis of the enzyme activity results of the liver samples showed that ( Fig. 9 ), the SOD levels of the COS group (chitooligosaccharides) and the FOS group (fructooligosaccharides) were higher than those of the control group, and there were no significant changes in other indicators; the T-AOC activity, CAT activity, GSH-Px and SOD activity of the WC group (Weissella sinusitis) and the CWC group (compound microecological preparation) were significantly higher than those of the other groups, and the effect of CWC was better, indicating that the antioxidant capacity of Weissella sinusitis was better than that of the other two probiotics, and the addition of compound microecological preparations could significantly improve the body's antioxidant capacity.

[0102] In addition, ACP in the nonspecific immune index is an acid hydrolase with a high content in lysosomes. It is considered to be a marker for identifying lysosomal subcellular components. Its increase is related to various diseases. ALP can be used as a natural anti-infective substance with bactericidal effect. Compared with the control group, the ACP activity and MDA enzyme activity of the CWC group were significantly reduced. Several other prebiotics and probiotics can also reduce the ACP level but have no significant effect on MDA activity, indicating that the use of microecological composite preparations can enhance the antibacterial effect of soft-shelled turtles, while there is no significant difference in ALP enzyme activity among several groups. Based on the above results, it can be concluded that the use of composite microecological preparations can further improve the antioxidant and antibacterial capabilities of soft-shelled turtles.

[0103] Example 6: Analysis of digestive enzyme activity

[0104] The intestines were immediately frozen in liquid nitrogen after dissection and stored in a -80°C refrigerator for later use. According to the manufacturer's instructions, the digestive enzyme activities of the intestinal tissues of the 8 groups of turtles collected in Example 3 after feeding were determined using a commercial kit (Nanjing Jiancheng Bioengineering Institute), including trypsin, lipase and amylase.

[0105] like Fig.10 As shown, compared with the control group, there was no significant difference in the activities of lipase and amylase in the six test groups. However, Weissella sinensis, Enterococcus faecalis and the compound probiotic preparation significantly increased the activity of trypsin, and the effect of Weissella sinensis was significantly superior to that of the other two groups.

[0106] Example 7: Analysis of Amino Acid Composition for Improving Muscle

[0107] Hitachi L8900 fully automatic amino acid analyzer was used to detect the changes in crude protein, essential amino acids, umami amino acids and four key amino acids in the hind leg muscles of the eight groups of soft-shelled turtles collected in Example 3 after feeding. The results of single-factor ANOVA were as follows: Fig.11 ), after adding chitosan oligosaccharide and composite probiotic preparation, the crude protein content in soft-shelled turtle muscle increased, and adding Lactococcus lactis could increase the essential amino acid content; adding chitosan oligosaccharide, oligofructose, Enterococcus faecalis, Weissella edodes or composite probiotic preparation could increase the umami amino acid content, but the effect of adding composite probiotic preparation was more significant, and at the same time, adding composite probiotic preparation could significantly increase the content of glutamic acid, aspartic acid and glycine. Therefore, composite probiotic preparation is preferred.

[0108] Example 8: Water quality adjustment analysis

[0109] Since prebiotics, probiotics or composite microecological preparations are added to the feed and then the feed is sprinkled and fed, the intestinal flora and water flora of the turtle are also affected by the feeding bacteria, and the water flora decomposes food residues or feces, etc., which will change the water quality. Some colonies will decompose substances to produce ammonia nitrogen, which is not conducive to the growth of the turtle. In order to explore the changes in water quality after feeding the 8 groups shown in Table 1 in Example 3, this embodiment uses a water quality analyzer (Green Carey) to measure water quality chemical oxygen demand, total phosphorus, nitrite nitrogen, ammonia nitrogen, and sulfide.

[0110] Chemical oxygen demand (COD) is a measure of the total amount of reducing substances (mainly organic matter) in water that can be oxidized by strong oxidants. When the COD value increases, it means that the water is polluted. Nitrite nitrogen and sulfur compounds are harmful substances in water, which will deteriorate water quality and affect the growth and health of aquaculture. Ammonia nitrogen can cause eutrophication of water and is the main oxygen-consuming pollutant in water. Excessive phosphorus content will cause excessive growth of algae, eutrophication of water, and algal blooms or red tides, disrupting the balance of the water. The higher the dissolved oxygen, the more favorable it is for aquatic animals to take in oxygen. Fig.12 As shown, compared with the control, after adding chitosan oligosaccharides alone to feed, the COD value, nitrite nitrogen and sulfur-containing compounds in the water body were significantly higher, and the dissolved oxygen level was reduced, indicating that the water quality was poor; after adding probiotics or compound microecological preparations to feed, the COD value, nitrite nitrogen and sulfur-containing compounds in the water body did not change significantly, but the ammonia nitrogen content of the compound microecological preparation group was significantly reduced, the dissolved oxygen content was higher, and the water quality was acidic, indicating that the water quality was better.

[0111] like Fig.13 and 14As shown in the figure, the abundance of Gemmatimonadota bacteria in CK was high. Studies have found that some gemmatimonadota species may produce harmful substances, such as toxins or pathogenic factors, causing infection or disease. At the phylum level, the abundance of Proteobacteria was reduced in COS, FOS and WC groups compared with the control group. This phylum contains many pathogens that may destroy the balance of intestinal microecology and induce intestinal infection, indicating that the addition of chitosan oligosaccharides, oligofructose or Weissella sinusitis can reduce the risk of soft-shelled turtles getting sick. The abundance of Bacteroidota was increased in COS, LL and CWC groups compared with the control group ( Fig.15 ), the study found that Bacteroidota can help break down food, especially complex carbohydrates, and can also form a protective biofilm in the intestine to help prevent the invasion of harmful microorganisms and strengthen the intestinal barrier function. Fig.16 ), the CK group was enriched with Terrisporobacter and Limnobacter, the COS group was enriched with Epulopiscium and OLB12, the FOS group was enriched with Mycobacterium, Flavobacterium, and Fluviicola, the WC group was enriched with Paeniclostridium, and the CWC group was enriched with Romboustsia, Turicibacter, and Terrimonas. Figure 17-18 As shown in the figure, the CWC group and the commercial protease group reduced the community diversity, indicating that the addition of probiotics to the feed of soft-shelled turtles also changed the bacterial community structure in the water.

[0112] Example 9: Comparison of composite microecological preparations with different components

[0113] According to the results of Examples 3 to 8, it can be seen that different prebiotics or probiotics have differences in intestinal flora composition, intestinal morphology, body immunity and digestion ability, and water quality regulation. Therefore, the composite microecological preparation composed of different prebiotics or probiotics should also have different comprehensive performance. In order to further compare the differences in composite microecological preparations prepared by different components, the best combination is screened out. In this embodiment, chitosan oligosaccharides and oligofructose are used as two prebiotics and three probiotics of Weissella edodes, Lactococcus lactis and Enterococcus faecalis are used to prepare four composite microecological preparations shown in Table 3, and the preparation method is the same as in Example 1. After the four composite microecological preparations are added to the feed, they are fed according to the method of Example 3, and the control is the basic diet. After that, the immune indicators in the liver tissue of the turtle are analyzed, and the analysis method is the same as in Example 5, and the results are shown in Tables 4 and 5.

[0114] Table 3 Formulas of different composite microecological preparations

[0115]

[0116] Table 4 Antioxidant index determination results

[0117]

[0118]

[0119] Table 5 Nonspecific immune index determination results

[0120]

[0121] As shown in Table 4 and Table 5, the four composite probiotic preparations had no significant effect on the activity of T-AOC and GSH-Px, and composite probiotic preparations 2, 3, and 4 had no significant promoting effect on the activity of five enzymes related to antioxidant effects and three enzymes related to nonspecific immunity, while composite probiotic preparation 1 could increase the activity of CAT, significantly increase the activity of SOD and reduce the level of MDA at the same time. At the same time, composite probiotic preparation 1 also reduced the level of ACP, indicating that adding composite probiotic preparation 1 to feed can significantly improve the antioxidant and antibacterial ability of soft-shelled turtles and improve the body's immunity. Therefore, the composite probiotic preparation prepared from chitosan oligosaccharides and Weissella sinus is preferred.

[0122] Example 10: Comparison of Weissella sinensis from different sources

[0123] Existing studies have isolated and obtained a variety of different sources of Weissella sinusoids, such as mandarin fish, etc. The effects of the composite probiotic preparations prepared from different Weissella sinusoids and chitosan oligosaccharides may be different. In order to further verify the difference between the composite probiotic preparations prepared from Weissella sinusoids from different sources, this embodiment uses Weissella sinusoids from mandarin fish and chicken to prepare composite probiotic preparations with chitosan oligosaccharides, and the preparation method is the same as that of Example 1. After these two composite probiotic preparations and the composite probiotic preparation 1 in Example 9 are added to the feed, they are fed according to the method of Example 3, and the control is the basic diet. After that, the immune indicators in the liver tissue of the turtle are analyzed, and the analysis method is the same as that of Example 5. The results showed that after feeding with the composite microecological preparation prepared from the sinus-eating Weissella from mandarin fish, the enzyme activities of T-AOC, CAT and GSH-Px were improved, but not significantly, the ALP level was reduced, and the water quality of the turtle breeding water body did not change significantly; after feeding with the composite microecological preparation prepared from the sinus-eating Weissella from chicken, the five indicators of antioxidant effect changed slightly, the ACP level was reduced, and the total phosphorus content and ammonia nitrogen content of the turtle breeding water body were reduced; and after feeding with the composite microecological preparation 1, the T-AOC, CAT and SOD activities were significantly increased, the MDA activity was reduced, the ACP and ALP levels were reduced, and the ammonia nitrogen content and chemical oxygen demand in the turtle breeding water body were significantly reduced, and the dissolved oxygen content was increased. Therefore, the composite microecological preparation provided by the present invention is preferred.

[0124] In summary, the above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, improvements and modifications can be made without departing from the core technology of the present invention, and these improvements and modifications should also fall within the scope of patent protection of the present invention. Any changes within the meaning and scope equivalent to the claims of the present invention should be considered to be included in the scope of the claims.

Claims

1. A feed for improving the water quality of turtle breeding water, characterized in that: The invention comprises Weissella sinensis, and the preservation number of the Weissella sinensis is CGMCC NO.32640.

2. The feed according to claim 1, characterized in that It also includes any one or more prebiotics selected from the group consisting of oligofructose, oligochitosan, and β-glucan.

3. The feed according to claim 1, characterized in that The prebiotic is chitosan oligosaccharide.

4. The feed according to claim 3, characterized in that The content of the Weissella sinusoidalis is 1×10 7 ~1×10 9 CFU / g, and the content of chitosan oligosaccharide is 0.1-0.5wt%.

5. A method for improving water quality when breeding soft-shelled turtles, characterized in that: Feed the soft-shelled turtle with the feed as described in any one of claims 1 to 4.

6. The method according to claim 5, characterized in that The feed is fed to the soft-shelled turtle in a splashing manner, and the feed enters the water body while being fed to the soft-shelled turtle. The water body flora decomposes the feed, and the probiotics or prebiotics in the feed regulate the water body flora to improve the water quality.

7. A use of feed for preparing a reagent for reducing the content of ammonia nitrogen, total phosphorus and chemical oxygen demand in turtle breeding water, characterized in that: The feed comprises Weissella sinensis, and the preservation number of the Weissella sinensis is CGMCCNO.32640.

8. A use of feed for preparing a reagent for increasing the dissolved oxygen content in turtle breeding water, characterized in that: The feed comprises Weissella sinensis, and the preservation number of the Weissella sinensis is CGMCCNO.32640.

9. A use of feed for preparing an agent for maintaining the balance of flora in turtle breeding water, characterized in that: The feed comprises Weissella sinensis, and the preservation number of the Weissella sinensis is CGMCCNO.32640.

10. The use according to claim 1, characterized in that The feed also includes any one or more prebiotics selected from the group consisting of oligofructose, oligochitosan and beta-glucan.

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